Production of rare ginsenosides by biotransformation of Panax notoginseng saponins using Aspergillus fumigatus

Panax notoginseng saponins (PNS) are the main active components of Panax notoginseng. But after oral administration, they need to be converted into rare ginsenosides by human gut microbiota and gastric juice before they can be readily absorbed into the bloodstream and exert their effects. The sources of rare ginsenosides are extremely limited in P. notoginseng and other medical plants, which hinders their application in functional foods and drugs. Therefore, the production of rare ginsenosides by the transformation of PNS using Aspergillus fumigatus was studied in this research. During 50 days at 25 ℃ and 150 rpm, A. fumigatus transformed PNS to 14 products (1–14). They were isolated by varied chromatographic methods, such as silica gel column chromatography, Rp-C18 reversed phase column chromatography, semi-preparative HPLC, Sephadex LH-20 gel column chromatography, and elucidated on the basis of their 1H-NMR, 13C-NMR and ESIMS spectroscopic data. Then, the transformed products (1–14) were isolated and identified as Rk3, Rh4, 20 (R)-Rh1, 20 (S)-Protopanaxatriol, C-K, 20 (R)-Rg3, 20 (S)-Rg3, 20 (S)-Rg2, 20 (R)-R2, Rk1, Rg5, 20 (S)-R2, 20 (R)-Rg2, and 20 (S)-I, respectively. In addition, all transformed products (1–14) were tested for their antimicrobial activity. Among them, compounds 5 (C-K) and 7 [20 (S)-Rg3] showed moderate antimicrobial activities against Staphylococcus aureus and Candida albicans with MIC values of 6.25, 1.25 μg/mL and 1.25, 25 μg/mL, respectively. This study lays the foundation for production of rare ginsenosides. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40643-024-00794-0.


Introduction
Panax notoginseng (Burk.)F.H. Chen is a well-known traditional Chinese medicine, which has various biological activities (Li et al. 2022a, b;Yang et al. 2020;Ye et al. 2010).P. notoginseng saponins (PNS) are the main active components of P. notoginseng.Among them, ginsenosides Rg 1 , Rb 1 , Rd, Re and notoginsenoside R 1 are the major saponins, accounting for over 80% of the PNS.Saponins have pharmacological effects, such as anti-inflammatory, antioxidant, inhibition of platelet aggregation, regulation of blood glucose and blood pressure, inhibition of neuronal apoptosis, protection of neurons, etc. (Duan et al. 2018;Lin et al. 2015;Wei et al. 2023;Xiong et al. 2019).However, the major ginsenosides in PNS (including Rg 1 , Rb 1 , Rd, Re, R 1 ) are difficult to be absorbed by the human body due to their high molecular weight, low membrane permeability, and low bioavailability (Cui et al. 2016;Upadhyaya et al. 2016).After oral administration, they need to be converted into minor ginsenosides by human gut microbiota and gastric juice before they can be readily absorbed into the bloodstream and exert their effects.In addition, pharmacological studies showed that rare saponins have better biological activity, but their content in P. notoginseng is very low (Park et al. 2010;Wei et al. 2011;Wu et al. 2012).Thus, a lot of studies have been focused on the conversion of major ginsenosides to rare ginsenosides (Li et al. 2022).Biotransformation is the most promising method to produce rare ginsenosides, which has the advantages of strong specificity, high yield, low cost and environmental friendliness (Zhang et al. 2023a, b;Li et al. 2022a, b).Studies showed that the genus Aspergillus has the ability of transformation saponins to rare ginsenosides.Such as Aspergillus tubingensis can convert ginsenoside Rb 1 , Rb 2 , Rc, and Rd to CK. (Song et al. 2023); Aspergillus Niger XD101 can convert ginsenoside Rb 1 to CK (Jiang et al. 2021).However, there have no reports on the separation, purification, and structural identification of PNS transformation products by the genus of Aspergillus.This study aimed to transform major ginsenosides into rare ginsenosides from PNS using fungus Aspergillus fumigatus, and to speculate their transformation pathways.This study reported for the first time the microbial conversion of PNS using A. fumigatus, and 14 rare ginsenosides were isolated from the converted products.This paper provides a new microbial conversion strain source for the large-scale preparation of rare ginsenosides, and also provides a theoretical basis for improving the medicinal value of P. notoginseng.

Strains
The strain of A. fumigatus was isolated from fresh P. notoginseng root soil in our previous research, and was conserved in Potato Dextrose Agar (PDA) medium.A voucher specimen (No.Yang20210907) was deposited at the Faculty of Life Science and Technology, Kunming University of Science and Technology.

Morphological observation of A. fumigatus
A. fumigatus was inoculated on PDA medium and cultured in 25 ℃ incubator for 3 days.Colony characteristics and morphological characteristics (under optical microscope) were observed.

Medium
PDA medium: potato extract powder 5 g/L, glucose 15 g/L, and agar 25 g/L.PDB medium: potato extract powder 5 g/L and glucose 15 g/L.

Microbial transformation of PNS by A. fumigatus
We transferred well-developed fungal hyphae from the surface of the agar slant to three 500 mL Erlenmeyer flasks containing 300 mL PDB medium.The cultures were grown for 3 d on a rotating shaker at 25 °C with shaking at 150 rpm to produce seed liquid.Then, the seed liquids were transferred to 500 mL reagent bottles that contained 300 mL of medium for expand fermentation.The cultures were then incubated using the same conditions as before.After 4 d, PNS (transformation substrate) were added to the cultures at the concentration of 5 mg/ mL.The cultures were incubated for additional 46 days at 25 °C with shaking at 150 rpm.Finally, A total of 172.5 g PNS was transformed and a total of 34.5 L fermentation broth were prepared.The mycelia were separated by filtration and the filtrate was extracted five times with n-butanol.The organic layer was concentrated under reduced pressure to afford a residue (161.5 g).

Antimicrobial activity
Transformation products (1-14) were evaluated for antimicrobial activity against 2 human pathogenic microbia.The tested microbia were: Staphylococcus aureus and Candida albicans.The activity of compounds 1-14 against human pathogenic microbia was tested by double dilution method.Compounds 1-14 and positive control ciprofloxacin were dissolved with DMSO and the concentration was 1 mg/mL.Pathogenic microbia were incubated in LB medium at 37 ℃ and 160 rpm for 8-12 h, microbia solution and compounds 1-14 were added to 96-well plates, incubated at 37 ℃, and the 96-well plate was observed to be clear and transparent without microbia growth after 12 h.The experiment was repeated three times.LB broth medium component: 20 g LB Broth dissolved in 1000 mL distilled water.

Morphological observation of A. fumigatus
A. fumigatum was inoculated on PDA and cultured in 25 ℃ incubator for 3 days.The following colony characteristics were observed: A. fumigatus is fluffy or flocculent, dark green (Fig. 1a).The conidial head is columnar, green when young, dark green when mature; The surface of the conidial stem is smooth, and many spores are scattered around it (Fig. 1b).

Conversion rate of four products of PNS
The conversion rate of substrates and the yield of products was defined as follows: Conversion rate (%) m: the mass of substrate; m 1 : the mass of remaining substrate; m 2 : the mass of products.
The major ginsenosides of PNS was notoginsenoside R 1 , ginsenosides Rg 1 , Re, and Rb 1 , the conversion rate of them were 32.52, 19.35, 24.74, and 100%, respectively.The conversion products and productivity were shown in Table 1.

Propose possible biosynthetic pathways of major ginsenosides Rg 1 , Re, Rb 1 and notoginsenoside R 1 of PNS
The transformation pathway of ginsenoside Rb 1 is proposed in Fig. S1A.Rb 1 obtained Rd after hydrolyzing the lateral glucose of C-20, so the two monomers share the same conversion pathway.The ginsenoside Rb 1 contains four glucopyranosyl moieties at the C-3 and C-20 position of aglycone.According to the isolated ginsenosides 20 (R/S)-Rg 3 , Rg 5 and Rk 1 , C-K, 20 (S)-I, the conversion pathway of Rb 1 can be predicted.The first pathway is the A. fumigatus attacked the outer β-(1 → 6)-glucosidic bond linkages to C-20 position of aglycone to produce Rd from Rb 1 , and was then followed by the hydrolysis of the outer β-(1 → 6)-glucosidic bond to C-3 position to produce F 2 , later followed by the hydrolysis of the inner β-(1 → 6)-glucosidic bond to C-3 position to produce CK or by heating and oxidizing the air to get 20 (S)-I.Another pathway was followed by the hydrolysis of the inner β-(1 → 6)-glucosidic bond to the C-20 position to produce 20 (R/S)-Rg 3 from Rd, then through dehydration reaction at the C-20 position to form a double bond with C-21 to get Rk 1 , or to form a double bond with C-22 to get Rg 5 .

Antimicrobial activity
The antimicrobial activities of compounds 1-14 against 2 pathogenic microbial were tested.As shown in Table 2, Compounds 5 and 7 have moderate antimicrobial activity against Staphylococcus aureus and Candida albicans, with MIC values of 6.25, 1.25 μg/mL and 1.25, 25 μg/mL, respectively.Additionally, compounds 10 and 11 also had certain antimicrobial activity against Staphylococcus aureus and Candida albicans.The antimicrobial activity of compounds 1-14 as shown in Table 2

Conclusion
Rare ginsenosides is a group of dammarane triterpenoids that exist in low natural abundance, which can be produced by physicochemical processing or metabolic transformation of major ginsenosides.Due to their small polarity and molecular weight, they exhibited potent biological activity comparing to the primary ginsenosides.The fungus A. fumigatus has the ability of transform PNS to rare ginsenosides.We isolated 14 rare ginsenosides from the transformation products.The structure analysis of 14 rare ginsenosides showed that they were the metabolites of ginsenosides Rb 1 , Rg 1 , Rd, Re and notoginsenoside R 1 (they are the major ginsenosides of PNS), respectively.Based on the structure of the transformation products, we speculate on the possible biological transformation pathways of saponins (Fig. S1).The conversion rates of four ginsenosides were calculated by HPLC analysis, and it was found that the conversion rates of ginsenosides Rb 1 , R 1 , Rg 1 , Re were 100, 32.52, 19.35, 24.74%, respectively.The yield of ginsenoside 20 (R)-Rg 3 , which has good anti-tumor effect, can reach 22.29%.The yield of other ginsenosides 20 (S)-Rg 3 , Rh 4 , 20 (S)-I, Rg 5 , Rk 3, Rk 1 and C-K could reach 17. 90, 6.42, 5.60, 6.30, 3.61, 3.25 and 0.19%, respectively.We found that the products of PNS converted by A.fumigatus were rich and varied, and the conversion rate of ginsenoside Rb 1 could reach 100%, which completely transformed components in PNS.
Our study found that the process of PNS transformed by A.fumigatus, involved a variety of reactions, including deglycosylation, dehydration, and oxygenation.So, we can obtain multiple products through transformation from PNS by this fungus.Compared with other studies (Song et al. 2023 andJiang et al. 2021), our transformation products are more abundant, we can not only obtain Rg 3 and CK as other literatures, but also can obtain many other rare ginsenosides (Rh 4 , 20 (S)-I, Rg 5 , Rk 3, Rk 1 , et al.).So, this study provided an active fungus to prepare diversity rare ginsenosides.Besides this, we deduced the transformation pathway of saponins, which can provide theoretical basis for the acquisition of target rare ginsenosides.
Through the transformation of PNS by A. fumigatus, this study can provide a method for obtaining the rare saponins, lay a foundation for the efficient utilization P. notoginseng, improve the pharmacological activity and economic value of saponins in P. notoginseng, and provide a basis and theoretical support for large-scale industrial preparation of rare saponins.

Fig. 1
Fig. 1 Morphology of A. fumigatus.a Colony morphology diagram; b Spore map of A. fumigatus Additional file1 Fig. S1.Proposed possible biosynthetic pathways of major ginsenosides Rg 1 , Re, Rb 1 and notoginsenoside R 1 of PNS by A. fumigatus.Fig. S2.HPLC analysis of transformation products of PNS by A. fumigatus.